ABSTRACT
[Objective] To investigate the prevalence, drug resistance and molecular characteristics of MRSA in hospital environment, so as to provide basis for prevention and control of nosocomial MRSA infection. [Methods]Specimens from Dec 2014 to May 2015 were collected from a tertiary hospital in Jing'an District of Shanghai and MRSA strains isolated from hospital environment and characterized by antimicrobial susceptibility testing and pulsed-field gel electrophoresis (PFGE). [Results]From 316 specimens were detected 46 MRSA strains with contamination rate being 14.6％ (46/316). And 22 (7.0％) were detected to be carriers of MRSA on the surfaces of ICU environment. MRSA had higher resistant rate to cefuroxime, levofloxacin, ciprofloxacin, Ampicillin/sulbactam, erythrocin, gentamycin than MSSA, and the difference was statistically significant (P<0.01). A total of 8 PFGE patterns were identified among the 14 MRSA isolates, which were mostly similar from one clone. [Conclusion] MRSA strains isolated from ICU in this hospital are multi-resistant to β-lactam antibiotics, quinolones and macrolides. There is clonal pollution in the environment. It is necessary to decrease the contamination rates between patients and environment, health care workers and environment, also it is worthwhile to increase the disinfection rate of environment.
ABSTRACT
ORF1 and ORF2 gene of porcine circovirus type 2 were cloned by PCR with the specific primers designed according to genome of PCV2 (AY035820). Following extraction and digestion, PCR products were subsequently inserted into universal transfer vector plECMV (deleted partial gE and gI of pseudorabies virus) to generate recombinant transfer plasmid pIEORF1-ORF2. The genomic DNA of PRV TK-/gE- /LacZ+ strain and pIEORF1-ORF2 were co-transfected into IBRS-2 cells with lipofectin, and recombinant virus TK- /gE- /gI- /ORF1-ORF2+ was selected by PCR with ORF1 gene and ORF2 gene primers respectively. The recombinant virus was analyzed with Southern blotting and Western blotting. The results indicated that ORF1 and ORF2 gene of PCV2 had been inserted into the genome of TK- /gE- /LacZ+ strain and the expressed ORF1-ORF2 fusion protein could react with PCV2 positive sera. Result of virus titers detection showed the insertion of ORF1 and ORF2 gene did not influence propagation of recombinant virus.
Subject(s)
Animals , Cell Line , Circovirus , Classification , Genetics , Gene Transfer Techniques , Genes, Viral , Herpesvirus 1, Suid , Genetics , Open Reading Frames , Genetics , Recombinant Proteins , Genetics , Recombination, Genetic , SwineABSTRACT
To produce the recombinant baculovirus transfer plasmid pFast-ORF2, the ORF2 gene of Porcine Circovirus type 2 (PCV2) was subcloned into baculovirus transfer vector (pFastBac(TM1) ) using Bac-to-Bac baculovirus expression system. E. coli DH10Bac (Gibco BRL) containing baculovirus shutter vector (bacmid) and helper vector was transformed with recombinant plasmid pFast-ORF2. Within E. coli DH10Bac, the ORF2 gene was transposed into the bacmid. The colonies of E. coli containing recombinant bacmid (Bac. ORF2) were collected by blue/white selection. The Bac. ORF2 was transfected into sf9 cells to yield AcNPV carrying the PCV2 ORF2 gene, referred to as Ac. ORF2. Expression of the ORF2 gene of PCV2 was confirmed by indirect immunofluorescent assay (IIFA), SDS-PAGE and Western-blotting. The expressed ORF2 gene product had a molecular mass of 28kD and could be recognized by the positive serum of PCV2. The results indicated the ORF2 gene was properly expressed in sf9 cell. It was noteworthy that many self-assembled virus-like particles (VLPs) were found in purified and phosphotungstic acid (PTA) stained PCV2 ORF2 protein by electron microscope. The particles were of similar morphology to the PCV2 virion and some self-assembled virus-like particles had darkly stained centers that made them appear to be empty capsids. Both PCV2 particles and self-assembled particles were approximately 17 nm in diameter.